title: Characteristics and requirements of basal autophagy in HEK 293 cells.
abstract: Basal autophagy-here defined as macroautophagic activity during cellular growth in normal medium containing amino acids and serum-appears to be highly active in many cell types and in animal tissues. Here we characterized this pathway in mammalian HEK 293 cells. First, we examined, side by side, three compounds that are widely used to reveal basal autophagy by blocking maturation of autophagosomes: bafilomycin A 1 (BafA1), chloroquine and vinblastine. Only BafA1 appeared to be without complicating side effects. Chloroquine partially inhibited mechanistic target of rapamycin (MTOR) activity, which would induce autophagy induction as well as block autophagosome maturation. Vinblastine caused the distribution of early omegasome components into punctate phagophore assembly sites, and therefore it would also induce autophagy, complicating interpretation. Basal autophagy was significantly sensitive to inhibition by wortmannin, and therefore required formation of phosphatidylinositol 3-phosphate (PtdIns3P), but it was twice as resistant to wortmannin as starvation-induced autophagy. We also determined that basal autophagy was significantly suppressed by MTOR activation brought about by overexpression of RHEB or activated RAGs. Finally we investigated the spatial relationship of nascent autophagosomes to the endoplasmic reticulum (ER) or to mitochondria by live imaging experiments under conditions that reveal basal autophagy (with BafA1 treatment), or upon MTOR inactivation (which would result in autophagy induction). Side-by-side comparison showed that under both basal and induced autophagy, 100% of autophagosomes first appeared in close proximity to ER strands. In parallel measurements, 40% were in close proximity to mitochondria under both conditions. We concluded that in HEK 293 cells, basal autophagy is mechanistically similar to that induced by MTOR inactivation in all aspects examined.
authors.affiliation: Signalling Programme; Babraham Institute; Babraham, Cambridge UK.
authors.forename: Patience
authors.initials: P
authors.lastname: Musiwaro
authors.forename: Matthew
authors.initials: M
authors.lastname: Smith
authors.forename: Maria
authors.initials: M
authors.lastname: Manifava
authors.forename: Simon A
authors.initials: SA
authors.lastname: Walker
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ktistakis
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D001343:Autophagy; D015415:Biomarkers / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D015415:Biomarkers; C477012:MAP1LC3A protein, human; D018942:Macrolides; D008869:Microtubule-Associated Proteins; D018129:Phosphatidylinositol Phosphates; C055525:phosphatidylinositol 3-phosphate; C040929:bafilomycin A1; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
keywords: autophagosomes; bafilomycin A1; basal autophagy; chloroquine; target of rapamycin; vinblastine